The pyruvate dehydrogenase complex (PDC) functions at an important branch point for energy metabolism in regard to the utilization and storage of cellular fuels--carbohydrate and fat. Mammalian PDC is a giant enzyme complex composed of six components with nine distinct subunits. Five subunits form an organized structure that allows them to carry out the coordinated series of reactions of the complex. Four of the subunits (two in a kinase and two in a phosphatase) function in the regulation of PDC by interconverting the pyruvate dehydrogenase (E1) component between an active (dephospho) form and inactive (phospho) form. Sixty transacetylase (E2) subunits assemble to form a polyhedral inner structure with the bulk of E2's mass in an extended, highly segmented outer structure consisting of 3 domains connected by relatively large and highly mobile linker regions. Two of these are lipoyl domains. The focus of this proposal is to understand the highly consequential roles of lipoyl domains in E1 and kinase function. The lipoyl domains serve as substrates of E1 in the rate limiting second step of PDC catalysis that generates a thioacetyl and reducing equivalents. The principal investigator will characterize how the lipoyl domains interact with E1 and will evaluate the likely prospect that phosphorylation blocks E1 catalysis by altering this lipoyl domain-E1 interaction. The kinase has been shown to be a member of a new eukaryotic family of kinases related to the prokaryotic histidine kinases. It binds tightly to the inner lipoyl domains of E2 by an unusual association involving the lipoyl prosthetic group. The proposed work focuses on the molecular mechanism by which the flexibly connected lipoyl domains of E2 operate both to facilitate enhanced kinase activity and to mediate effector modulation of kinase activity. Studies are designed to explain enhanced function by dissecting the mechanisms whereby tightly bound kinase rapidly moves between lipoyl domains and otherwise has its activity enhanced when bound just to an isolated lipoyl domain. The principal investigator will also investigate how a specialized signal translation mechanism functions to establish feedback control of mammalian PDC through a lipoyl prosthetic group-mediated process that stimulates kinase activity. To discriminate the multiple roles played by lipoyl domains in E1 and kinase function, the principal investigator will employ native, mutated, and prosthetic group modified lipoyl domains as free, carrier-linked, and oligomeric E2 structures. Understanding how PDC is regulated constitutes essential knowledge for diagnosing human diseases. Aberrant PDC operation is associated with diabetes and obesity. Inborn errors in components cause severe defects. Primary biliary cirrhosis is an autoimmune disease with antibodies to E2 and other PDC subunits.